IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/08/05

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Testing AHL degradation by aiiA

  • The product of the aiiA gene AHL-lactonase degrades AHL in many strains of Bacillus, however, it is unclear to what extent (if any) B. subtilis strain 168 degrades AHL. If 168 is good at degrading AHL this will be a problem when our signaling system is eventually moved into strain 168.

We are testing B. subtilis strain 168’s ability to degrade AHL by exposing synthetic AHL to the strain and then testing its ability to be recognized by the receiver.

  • Materials

Bacillus subtilis - protease deficient (NPR and APR) derived from strain 168 - grown up in an over night culture of LB.

Synthetic AHL – Sigma K3007 – AHL powder was dissolved in EA buffer and diluted to 10uM concentration

E. coli with T9002 AHL receiver grown up in overnight culture of LB

All plates contain soft agar with 200uL of E. coli AHL receiver mixed-in. Plates 1,2 and 4 contain Bacillus in fresh LB medium, while plate 5 contains Bacillus in it’s overnight LB. Plate 5 was used to test if an excreted product of Bacillus is responsible for the degradation of AHL. The product of the aiiA gene in Bacillus subtilis is most likely not exported.

  • Plate 1: 10uL Bacillus + 10uL AHL
  • Plate 2: 10uL Bacillus + 10uL EA buffer (negative control + does EA buffer kill bacillus?)
  • Plate 3: 10uL LB + 10uL AHL (positive control)
  • Plate 4: 10uL Bacillus + 10uL AHL incubated together for 1.5 hours prior to plating
  • Plate 5: 10uL Bacillus in overnight LB + 10uL AHL
  • Results

plates visualized 3 hours after AHL/Bacillus inoculation.....

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